RESUMO
To facilitate the N-terminal tagging of essential genes at their genomic locus and under control of their own promoters we have developed a series of novel polymerase chain reaction templates. Initially, a 1.8 kb DNA fragment is integrated upstream of the ATG of the gene of interest. This fragment encodes the tag, a loxP site, a selectable marker, an exogenous nmt1 promoter and a second loxP site. In a single homologous integration event, the gene of interest is placed under control of the thiamine regulated nmt1 promoter, allowing identification of potential integrants on the basis of phenotype. Subsequently, this integrant strain is transformed with a plasmid expressing the Cre recombinase. This results in excision of the marker and nmt1 promoter and leaves sequences encoding an in-frame tag at the N-terminus of the gene of interest under the control of its native promoter. We have created TAP-cdc22, TAP-suc22 and TAP-rad50 strains using this N-tagging system, and developed a range of vectors for introducing TAP-, (His)10HA-, (His)6Myc- and EGFP.
Assuntos
Cromossomos Fúngicos/genética , Genes Essenciais/genética , Genes Fúngicos/genética , Integrases/genética , Schizosaccharomyces/genética , Proteínas Virais/genética , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação/genética , Western Blotting , Proteínas de Ciclo Celular/genética , Integrases/metabolismo , Dados de Sequência Molecular , Plasmídeos/genética , Recombinação Genética , Ribonucleotídeo Redutases/genética , Ribonucleotídeo Redutases/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Transformação Genética , Proteínas Virais/metabolismoRESUMO
In order to gain a more global view of the activity of histone demethylases, we report here genome-wide studies of the fission yeast SWIRM and polyamine oxidase (PAO) domain homologues of mammalian LSD1. Consistent with previous work we find that the two S. pombe proteins, which we name Swm1 and Swm2 (after SWIRM1 and SWIRM2), associate together in a complex. However, we find that this complex specifically demethylates lysine 9 in histone H3 (H3K9) and both up- and down-regulates expression of different groups of genes. Using chromatin-immunoprecipitation, to isolate fragments of chromatin containing either H3K4me2 or H3K9me2, and DNA microarray analysis (ChIP-chip), we have studied genome-wide changes in patterns of histone methylation, and their correlation with gene expression, upon deletion of the swm1(+) gene. Using hyper-geometric probability comparisons we uncover genetic links between lysine-specific demethylases, the histone deacetylase Clr6, and the chromatin remodeller Hrp1. The data presented here demonstrate that in fission yeast the SWIRM/PAO domain proteins Swm1 and Swm2 are associated in complexes that can remove methyl groups from lysine 9 methylated histone H3. In vitro, we show that bacterially expressed Swm1 also possesses lysine 9 demethylase activity. In vivo, loss of Swm1 increases the global levels of both H3K9me2 and H3K4me2. A significant accumulation of H3K4me2 is observed at genes that are up-regulated in a swm1 deletion strain. In addition, H3K9me2 accumulates at some genes known to be direct Swm1/2 targets that are down-regulated in the swm1Delta strain. The in vivo data indicate that Swm1 acts in concert with the HDAC Clr6 and the chromatin remodeller Hrp1 to repress gene expression. In addition, our in vitro analyses suggest that the H3K9 demethylase activity requires an unidentified post-translational modification to allow it to act. Thus, our results highlight complex interactions between histone demethylase, deacetylase and chromatin remodelling activities in the regulation of gene expression.